1. Field of the Invention
This invention relates generally to antibody variants. In particular, antibody variants of parent antibodies are disclosed which have one or more amino acids alterations respect to the parent antibody and a binding affinity for toxin Cn2 which is at least about 10.5 fold stronger than the binding affinity of the parent antibody for the toxin. In another embodiment the invention relates to antibody variants that neutralizes the lethal effect of both the Cn2 toxin and the whole C. noxius venom. The invention is also related to the coding DNAs for the antibody variants; to vector molecules comprising said coding DNAs, to cells comprising said vectors and methods for the production of the antibodies. The invention relates too to solid phases comprising the antibody variants adhered and to diagnostic systems to detect the presence of toxin Cn2 in samples, comprising immunodiagnostic systems like ELISA and immunochromatographic assay which comprise the antibodies of the present invention. Additionally, the present invention relates to a method to select improved antibodies from a mutagenized library, where said antibodies are improved not only in its affinity but in its stability too.
2. Background Art
Antibodies are proteins, which exhibit binding specificity to a specific antigen. Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called (alpha), (delta), ε(epsilon), γ(gamma), μ(mu), respectively. Of the various human immunoglobulin classes, only human IgG1, IgG2, IgG3 and IgM are known to activate complement.
It was disclosed in patent application WO92/01047 that antibody fragments can be displayed on the surface of bacteriophage and that they will bind antigen. Antibody fragments can be directly selected using this characteristic. This ability to isolate antibody fragments (F(ab′)2, Fab, Fv, scFv and VH) using their display on the surface of filamentous bacteriophage has opened up the prospect of the isolation of antibody specificities (i.e. antibodies directed against a particular antigen) that were difficult or impossible to isolate previously. In particular WO92/01047 demonstrates that antibody specificities can be isolated from a human who has not been specifically immunized (‘un-immunized’), even specificities for antigens such as 2-phenyl-5-oxazolone to which humans will not normally be exposed.
In vivo, affinity maturation of antibodies is driven by antigen selection of higher affinity antibody variants which are made primarily by somatic hypermutagenesis. A “repertoire shift” also often occurs in which the predominant germline genes of the secondary or tertiary response are seen to differ from those of the primary or secondary response.
Various research groups have attempted to mimic the affinity maturation process of the immune system, by introducing mutations into antibody VH and VL genes in vitro and using affinity selection to isolate mutants with improved affinity. Such mutant antibodies can be displayed on the surface of filamentous bacteriophage and improved antibodies can be selected by their better affinity for antigen or by their kinetics of dissociation (off-rate) from antigen.
Scorpionism
Scorpion stings in Mexico reach over 200,000 accidents per year with a mortality of approximately 700 people during the decades of the seventies and eighties. For the nineties, the reported fatalities were 300 and by 1998, 136 persons. During 2002 the fatal cases diminished to 70 (Weekly Epidemiological Bulletin, Mexican Health Ministry). The decrement on the mortality rate coincided with a National Campaign for anti-venom utilization, sponsored by the Mexican Institute of Social Security. Serotherapy (heterologous immune serum administration), has been used during the last century for treatment of poisonings caused by animal bites and stings in humans (Choumet, V., Audebert, F., Riviere, G., Sorkine, M., Urtizberea, M., Sabouraud, A., Scherrmann, J. M. & Bon, C. (1996) Adv Exp Med Biol 391, 515-20). The antivenom which is currently used in Mexico consists of purified bivalent F(ab′)2 fragments obtained by hyper-immunizing horses with a water extract from venomous glands of Centruroides scorpions (Calderon-Aranda, E. S., Hozbor, D. & Possani, L. D. (1993) Toxicon 31, 327-37). Polyclonal antibodies present in horse serum, are raised against the total components of the venom, however only a reduced number of toxic components are important for poisoning. From 221 species living in México, only 8 are dangerous to human beings (Dehesa-Davila, M. (1989) Toxicon 27, 281-6). The venom from different species of scorpions of the genus Centruroides are very similar in terms of toxic components (Possani, L. D., Becerril, B., Delepierre, M. & Tytgat, J. (1999) Eur J Biochem 264, 287-300). It is worth mentioning that in scorpion venoms there are short and long-chain peptides, known to be specifically toxic to mammals. The deadly effect was demonstrated to be for their effect on target molecules known as ion-channels. There are several distinct ion-channels that preside the permeability of many ions, such as: Na+, K+, Ca2+, Cl−. These are integral-membrane proteins that control cellular excitability. The most important toxins from scorpion venoms are those that recognize sodium channels (Possani, L. D., Becerril, B., Delepierre, M. & Tytgat, J. (1999) Eur J Biochem 264, 287-300). Thus, the identification of the deadly components (mainly toxins specific for sodium channels) in those 8 venoms could help to obtain neutralizing recombinant antibodies, which would be the constituents of the next generation of antisera. A more specific antiserum would result in a safer medicine in terms of the reduced number of distinct antibodies present and the use of homologous human antibodies, replacing the horse antibodies presently used.
BCF2, a murine monoclonal antibody characterized in our laboratory (Zamudio, F., Saavedra, R., Martin, B. M., Gurrola-Briones, G., Herion, P. & Possani, L. D. (1992) Eur J Biochem 204, 281-92), neutralizes the toxic effects of Cn2 (a toxin specific for sodium channels of mammals), one of the most abundant and toxic components of the venom from the scorpion Centruroides noxius Hoffmann (6.8% of total venom; LD50=0.25 μg/20 g of mouse weight). BCF2 is also able of neutralizing the whole venom (LD50=2.5 μg/20 g of mouse weight) (Licea, A. F., Becerril, B. & Possani, L. D. (1996) Toxicon 34, 843-7). These findings suggested the possibility of obtaining recombinant antivenom of human origin. It would consist exclusively of specific antibody fragments, would be autologous, safer and more efficient for therapeutic application in humans.
The expression of several antibody formats on the surface of filamentous phages (phage display), has allowed the generation of large repertoires for different purposes, revolutionizing among others, the field of antibody engineering (Stockwin, L. H. & Holmes, S. (2003) Biochem Soc Trans 31, 433-6., Brekke, O. H. & Loset, G. A. (2003) Curr Opin Pharmacol 3, 544-50., Benhar, I. (2001) Biotechnol Adv 19, 1-33., Roque, A. C., Lowe, C. R. & Taipa, M. A. (2004) Biotechnol Prog 20, 639-54). The panning of these repertoires with different antigens constitutes a selection step analogous to that occurring in the immune system (Hoogenboom, H. R. & Winter, G. (1992) J Mol Biol 227, 381-8., Winter, G., Griffiths, A. D., Hawkins, R. E. & Hoogenboom, H. R. (1994) Annu Rev Immunol 12, 433-55), which allows the isolation of antibody fragments of diverse specificities.